Composition comprising phytosphingosine derivatives for apoptosis induction

ABSTRACT

The present invention is related to compositions for the induction of apoptosis containing phytosphingosine derivatives as effective components. The present invention is related to compositions for the induction of apoptosis containing Vit D 3  or calcipotriol as an effective component in addition to phytosphingosine derivatives. The compositions of the present invention include pharmaceutical compositions or cosmetic compositions having an activity to induce apoptosis. The present invention offers a method of prevention or treatment of various skin diseases, various tumors, various cancers, etc. that may be prevented or cured by the induction of the activity of apoptosis in living bodies, which is comprised of the steps of administration of compositions for the induction of apoptosis containing phytosphingosine derivatives as effective components and irradiation of UVB to psoriatic lesions. Therefore, the compositions of the present invention are useful for the prevention or treatment of various skin diseases, various tumors, various cancers, etc. that may be prevented or cured by the induction of the activity of apoptosis in living bodies.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is related to compositions containingphytosphingosine derivatives for the induction of apoptosis.

2. Description of the Prior Art

Apoptosis refers to a programmed cell death, which is one mode of celldeath occurring under the physiological and pathological conditions.

It has been known that while the number of cells is maintainedconstantly in normal tissues since the proliferation of cells andapoptosis are balanced, cancer cells are proliferated in tumoroustissues since the number of cells is increased due to inadequateapoptosis compared to a rapid proliferation of cells (Raff, M. C.,Nature, 356:397, 1992). The factors related to the induction ofapoptosis are known to include p53, bcl-2, bcl-XL, caspase, etc.(Wyllie, A., Nature, 389:237, 1997). Once the apoptotic program isactivated, programmed cell death starts with blebbing of the membrane,followed by degradation of the chromosomal DNA by nucleases, resultingin condensation and fragmentation of DNA Apoptosis plays an importantrole in the generation of fetuses and functions of the skin, internalorgans, and immunologic organs.

Inflammatory skin diseases are caused by the interaction of many immunecells including lymphocytes with keratinocytes occupying most of skincells, as immune cells are penetrated into the skin ultimately. Thesekeratinocytes affect on the proliferation of immune cells by secretingmany cytokines participating in immune functions, and are supplied withmany factors involved in the proliferation of the keratinocytes fromimmune cells. These cells and surrounding lymph nodes are calledskin-associated lymphoid tissues (SALT). In view of this, the skin isregarded to be not only the protective membrane of our bodies simply butalso one of immune organs.

Among skin diseases related to disregulation of apoptosis, psoriasis isa disease characterized by hyperproliferation of keratinocytes, andinfiltration and activation of various inflammatory cells, inparticular, T-cell. Since keratinocytes in psoriatic patients highlyproliferate along with angiogenesis, compounds inducing apoptosis inpsoriatic patients are suggested to be effective drugs for themanagements of psoriasis.

Particularly, it is known that ceramide, a sphingolipid derivative,converted from sphingomyelin by SMase (sphingomyelinase) activated bystimuli such as tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1),interferon-γ (IFN-γ), FAS ligand, and irradiation, acts as a secondmessenger to mediate cell differentiation, inhibition of cell cycle,proliferation and apoptosis.

Also, it is known that vitamin D₃ or calcipotriol induces apoptosis andcytotoxicity to hyperproliferative keratinocytes.

Further, it has been reported that apoptosis is induced by Smase whichis activated by the irradiation of UV light and accelerates formation ofceramide in the cells. Still further, it is known that growth factorsand cytokine surface receptors activated by irradiation of UV lightstimulate cytokine or growth factor signal transduction pathway.

UV light is divided into UVA (200-290 nm), UVB (290-320 nm), and UVC(320-400 nm) according to the wavelength. It has been reported thatthese facilitate immune suppression functions and cause apoptosis invivo or in culture cells.

The inventors of the present invention completed the present inventionas they found that the effect of induction of apoptosis was shownsignificantly in a specific phytosphingosine derivative while theysearched for effective compounds for various diseases showing thepreventive or treatment effects through induction of the apoptosis.

Proposed in the present invention are compositions for inducingapoptosis having phytosphingosine derivatives as effective components.

SUMMARY OF THE INVENTION

In the present invention, compositions inducing apoptosis containingphytosphingosine derivatives as effective components are provided for.

In addition to phytosphingosine derivatives, in the present invention, acomposition inducing apoptosis containing vitamin D₃ or calcipotriol asan effective component is suggested.

The compositions of the present invention are characterized by that thephytosphingosine derivative is one or more compounds selected from agroup comprised of phytosphingosine (PS), phytosphingosine-HCl (PS-HCl),C6-phytosphingosine (C6-PS), CLA-phytosphingosine (CLA-PS), tetraacetylphytosphingosine (TAPS), and N-acetyl phytosphingosine (NAPS).

The compositions of the present invention include pharmaceutical orcosmetic compositions having the activity to induce apoptosis.

Proposed in the present invention is a method of prevention or treatmentof various skin diseases, tumors, cancers, etc. that may be prevented ortreated by inducing apoptosis in vivo comprised of the steps ofadministration of a composition for inducing apoptosis containingphytosphingosine derivatives as effective components and of irradiatingUVB to psoriatic lesions.

The above preventive or treatment method is characterized by that aphytosphingosine derivative is one or more compounds selected from agroup comprised of phytosphingosine, phytosphingosine-HCl,C6-phytosphingosine, CLA-phytosphingosine, tetraacetyl phytosphingosine,and N-acetyl phytosphingosine.

The compositions of the present invention show the cytotoxic effect inall of human keratinocyte cell line, human skin cancer cell line, humanumbilical vein endothelial cells and peripheral blood mononuclear cells.

Among the compositions of the present invention, those having TAPS andNAPS as effective components show a significant apoptosis effect in thehuman keratinocyte cell line.

Among the compositions of the present invention, those containing TAPS,NAPS and PS as effective components have an effect of suppressing theactivity of Th1 cells that are related to inducing of psoriasis,particularly.

Among the compositions of the present invention, those containingvitamin D₃ or calcipotriol, that is used for an effective treatmentagent of psoriasis, as an additional effective component, show asignificantly increased apoptosis effect compared to other compositionsof the present invention containing a phytosphingosine derivativesingly.

If the compositions of the present invention are administered and UVB isirradiated, they show a significantly increased apoptosis effect thanwhen a phytosphingosine derivative is administered singly.

The dosage of irradiation of UVB in the method of treatment of psoriasisof the present invention when the compositions of the present inventionare administered is 50 mJ/cm²-2 J/cm².

The proteins involved in inducing apoptosis by the compositions of thepresent invention are caspase-3, p53, Chk1, etc.

Caspase-3 is cleavaged to the active form the maximum 3 hours after thecomposition of the present invention is treated and shows a remarkableability to induce apoptosis, etc.

The compositions of the present invention is useful for the preventionor treatment of various skin diseases, tumors, cancers, etc. that may beprevented or treated by inducing apoptosis in vivo.

Concretely, the diseases that may be prevented or cured by thecompositions of the present invention include abnormal skin diseasessuch as eczema, psoriasis, ichthyosis, etc.; skin diseases such asatopic dermatitis, dermatitis, itching, microbism, acne, wound, etc.;abnormal skin diseases induced by a long-time exposure to UV light anddermal aging; skin cancer, etc.

Particularly, the compositions of the present invention are useful forthe prevention and treatment of keratinization diseases such aspsoriasis, ichthyosis, etc.; abnormal skin diseases induced by along-time exposure to UV light and dermal aging; and skin cancer.

The compositions of the present invention may additionally contain oneor more effective components showing the same or similar functions.

Further, the compositions of the present invention may additionallycontain one or more effective components showing other functions.

Still further, the compositions of the present invention may include oneor more carriers that are allowable pharmacologically in addition toeffective components described in the above. Saline solution, sterilizedwater, Ringer's solution, buffered saline solution, dextrose solution,malto dextrine solution, glycerol, ethanol, and a mixture of one or moreof the above may be used for pharmacologically allowable carriers, andif necessary, other usual additives such as an anti-oxidant, buffersolution, bacteriostatic, etc. may be added. Also, a diluent, dispersionagent, surfactant, binder, and lubricant may be added and formulatedadditionally. And further, it may be formulated desirably according toeach disease or component by using a proper method in the present fieldor a method disclosed in Remington's Pharmaceutical Science (updatedversion), Mack Publishing Company, Easton, Pa.

It is desirable to administer the compositions of the present inventionlocally, and they may be offered in the form of an ointment, cream,emulsion, plaster, powder, impregnated pad, solution, gel, spray,lotion, or suspension.

A phytosphingosine derivative occupies 0.05-10.0 weight %, preferably0.1-5.0 weight % of the total weight of the composition of the presentinvention. And the compositions of the present invention are applied tothe psoriatic lesion a few times a day at a dose of about 10-30 ml or10-30 g.

A cosmetic composition of the present invention is not particularlylimited to its type of formulation. That is, it may be in the form of atender lotion, astringent, nutritious lotion, eye cream, nutritiouscream, massage cream, cleansing cream, cleansing foam, cleansing water,powder, essence, pack, emulsion, lotion, ointment, gel, polymeric orlipid vesicle or nanosphere or microsphere, soap or shampoo, etc. And inthe cosmetic composition of each formulation, a person skilled in theart may select and blend components other than phytosphingosinederivatives according to the type of formulation, purpose of use, etc.of other cosmetics.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, aspects and advantages will be betterunderstood from the following detailed description of a preferredembodiment of the invention with reference to the drawings, in which:

FIG. 1 shows graphs showing the cytotoxic effect of phytosphingosinederivatives which are effective components of the composition of thepresent invention in a dose dependent manner;

FIG. 2 shows graphs in which the effects of the compositions of thepresent invention on the cytotoxicity in immune cells separated fromspleen of mice are observed;

FIG. 3 shows graphs in which the effects of the compositions of thepresent invention on the cytotoxicity of peripheral blood mononuclearcells (PBMC) from human are observed;

FIG. 4 shows graphs in which the effects of the compositions of thepresent invention on the activity of human Th1 cells by the mixedleucocyte reaction are observed;

FIG. 5 shows graphs in which the effects of the compositions of thepresent invention on the activity of human Th1 cells by the reactionamong allogenic cells are observed.

FIG. 6 shows diagrams showing the effect of the compositions of thepresent invention on the apoptosis by the TUNEL assay;

FIG. 7 shows diagrams showing the effect of NAPS and TAPS which areeffective components of the compositions of the present invention on theapoptosis in a time dependent manner at a concentration of 30 μM;

FIG. 8 is a diagram showing the effect of TAPS which is an effectivecomponent of the compositions of the present invention on cell cycleusing FACS;

FIG. 9 is a diagram in which the effect of TAPS which is an effectivecomponent of the compositions of the present invention on the mitosis isobserved;

FIG. 10 is a diagram in which genes involved in apoptosis induced byTAPS which is an effective component of the compositions of the presentinvention is observed;

FIG. 11 is a diagram in which the effect of NAPS and TAPS which iseffective components of the compositions of the present invention on theincrease of the cleavaged active caspase-3;

FIG. 12 is a diagram showing the expression of p53 and Chk1 proteins ata concentration of 30 μM of TAPS which is an effective component of thecompositions of the present invention;

FIG. 13 is a diagram showing the expression of Chk1 protein according tothe time of NAPS and TAPS which are effective components of thecompositions of the present invention; and

FIG. 14 is a diagram in which the effects of treatment with NAPS, aneffective component of the compositions of the present invention, 5-7days after it is applied to the psoriatic lesion are observed.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT OF THE INVENTION

Hereinafter, a preferred embodiment of the present invention ispresented in order to assist understanding of the present invention.

Firstly, the cytotoxicity is measured by the MTT assay. Phytosphingosine(PS), phytosphingosine-HCl (PS-HCl), C6-phytosphingosine (C6-PS),CLA-phytosphingosine (CLA-PS), tetraacetyl phytosphingosine (TAPS), andN-acetyl phytosphingosine (NAPS) are dissolved into DMSO to have thefinal concentration of 1-100 μM.

1. Effects of the Compositions of the Present Invention on theCytotoxicity in the Human Keratinocyte Cell Line

In order to study the effects of the compositions of the presentinvention on the cytotoxicity in the human keratinocyte cell line HaCaTcells, the cell viability is analyzed by the MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay.HaCaT cells are supplied by Professor N. Fuseng of German CancerResearch in Germany. HaCaT cells are seeded in a density of 1×10⁶ cellsin a 100-nm dish and cultured in DMEM (Dulbecco's Modified Eagle Medium)containing 10% fetal bovine serum (FBS, GIBCO), 100 units/ml penicillin,and 100 μg/ml streptomycin for 48 hours. They are treated with trypsinand 1-2×10⁴ cells per well are seeded again in a 96-well plate by usinga serum-free culture medium, about 3 hours after which they are culturedby treatment with phytosphingosine derivatives. After they are culturedfor 24 hours, the MTT reagent is added at a concentration of 2 mg/ml,cultured for 4 hours, suspended in DMSO after removing the culturemedium completely in order to measure O.D. at 540 nm. C2-ceramide isused for comparison. The results of measurement are shown in Table 1 andFIG. 1 as follows: TABLE 1 Cell viability of phytosphingosinederivatives versus concentration (1-30 μM) PS PS-HCl C6-PS CLA-PS NAPSTAPS C2-ceramide con 100 ± 6.2  100 ± 9.6 100 ± 7.3  100 ± 0.6  100 ±8.6  100 ± 1.2  100 ± 2.0   1 μM 97 ± 11.1 100 ± 4.1  84 ± 16.8 66 ± 7.8103 ± 6.9  94 ± 0.1 97 ± 3.8  3 μM 93 ± 3   101 ± 2.2 96 ± 7.2 64 ± 9.999 ± 3.5 93 ± 2.9 98 ± 2.5 10 μM 86 ± 10.1  79 ± 7.0  74 ± 10.1 58 ± 0.486 ± 2.1 76 ± 1.4 75 ± 1.3 30 μM 51 ± 9.6   36 ± 6.8 87 ± 1.3 55 ± 9.117 ± 0.8 17 ± 1.1 33 ± 0.6

As shown in Table 1 and FIG. 1, all of 6 kinds of phytosphingosinederivatives of the present invention suppresses proliferation of cells.It is seen particularly that NAPS and TAPS have a more superiorcytotoxic effect than C2-ceramide has by 16% as C2-ceramide shows a 67%cytotoxicity while NAPS and TAPS show a 83% cytotoxicity at 30 μM.

2. Effects of the Compositions of the Present Invention on theCytotoxicity in the Human Skin Cancer Cell Line (A431)

In order to study the effects of the compositions of the presentinvention on the cytotoxicity in A431 cells, human skin cancer cellline, the MTT assay is used. The method of cell culture and MTT assayare the same as those in the above method. C2-ceramide is used forcomparison. The results of measurement are shown in Table 2 below: TABLE2 Cell viability of phytosphingosine derivatives versus concentration(3-50 μM) PS PS-HCl C6-PS CLA-PS NAPS TAPS C2-ceramide con 100 ± 10.8  100 ± 10.8 100 ± 10.8 100 ± 10.8 100 ± 10.8 100 ± 10.8 100 ± 10.8  3 μM85 ± 10.7 105 ± 5.8 106 ± 9.7   91 ± 11.0 100 ± 10.2 113 ± 11.2 113 ±19.8 10 μM 30 ± 1.9  109 ± 8.6 61 ± 8.1  98 ± 12.9 75 ± 6.9  83 ± 13.9 97 ± 14.7 30 μM 2 ± 0.1  7 ± 1.6  57 ± 10.4 60 ± 6.2  31 ± 10.0 12 ±1.7 82 ± 1.4 50 μM 2 ± 0.2  2 ± 0.2 44 ± 9.6 65 ± 5.5  1 ± 0.2  5 ± 1.2 74 ± 12.8

As shown in Table 2, it is seen that PS, PS-HCl, NAPS, and TAPS have amore superior cytotoxic effect than that of C2-ceramide by 69-72% asC2-ceramide shows a 26% cytotoxicity while PS, PS-HCl, NAPS, and TAPSshow a 95-98% cytotoxicity at 50 μM.

3. Effects of the Compositions of the Present Invention on theCytotoxicity in Human Umbilical Vein Endothelial Cells (HUVEC)

The MTT assay is used for the above purpose. Primary human umbilicalvein endothelial cells are cultured from the human umbilical vein. Themethod of cell culture and the MTT assay are the same as those in theabove. C2-ceramide is used for comparison. The results of measurementare shown in Table 3 below: TABLE 3 Cell viability of phytosphingosinederivatives versus concentration (3.5-30 μM) PS NAPS TAPS C2-ceramidecon 100 ± 10.6 100 ± 8.5  100 ± 10.1 100 ± 15.7 3.5 μM  64 ± 4.7 66 ±7.5 47 ± 8.4  90 ± 11.6  7 μM 19 ± 1.8  51 ± 10.5 22 ± 1.9 60 ± 7.7 15μM 14 ± 0.9 19 ± 3.4 15 ± 0.7 61 ± 8.4 30 μM 13 ± 1.0 14 ± 0.6  9 ± 0.534 ± 2.3

As shown in Table 3, it is seen that PS, NAPS, and TAPS have a moresuperior cytotoxic effect than that of C2-ceramide by 20-25% asC2-ceramide shows a 66% cytotoxicity while PS, NAPS, and TAPS show a86-91% cytotoxicity at 30 μM.

4. Effects of the Compositions of the Present Invention on theCytotoxicity in Immune Cells Separated from Spleen of Mice

The effect of the composition of the present invention on thecytotoxicity in immune cells (T cells, B cells, macrophages, monocytes,etc.) was examined.

The spleen was removed from normal mice (BDF) and single cells wereobtained from the spleen by mechanical aggregation method. Andmononuclear cells containing T cells, B cells, macrophages and monocyteswere isolated from single cells by density gradient centrifugationmethod using Ficoll Hypaque. Briefly, single cells were treated with PBSand overlayed on Ficoll Hypaque and centrifuged at 1500 rpm for 30 min.After centrifugation, the cells around the boundary of theFicoll-Hapaque were collected and washed three times with PBS.

The isolated cells were cultured for 24 hours in the presence of 1, 10,50 and 100 μM of NAPS, TAPS, and PS to examine the effect ofcytotoxicity, i.e. the degree of proliferation of the cells.

C2-ceramide was used as control. The medium used in this experiment wasRPMI 1640 suplemented with 10% of FBS and 100 μg/ml of penicillin andstreptomycin.

The effect of cytotoxicity was measured by MTT-assay as shown in FIG. 2.

The results in FIG. 2 show that IC₅₀ of C2-ceramide, NAPS, and PS wasabout 75 μM, and that of TAPS was 100 μM (IC₅₀ means the concentrationthat has the half effect.). The result shows that the compositions ofthe present invention have the cytotoxicity to immune cells obtainedfrom spleen of mice.

5. Effects of the Compositions of the Present Invention on theCytotoxiciy in Peripheral Blood Mononuclear Cells (PBMC) from HumanBlood

The effects of the composition of the present invention on thecytotoxicity in immune cells (T cells, B cells, macrophages, monocytes,etc.) obtained from human blood were examined.

The blood was obtained from healthy volunteers and PBMC from the bloodby mechanical aggregation method. And mononuclear cells containing Tcells, B cells, macrophages and monocytes were isolated from singlecells by density gradient centrifugation method using Ficoll Hypaque.Briefly, single cells were treated with PBS and overlayed on FicollHypaque and centrifuged at 1500 rpm for 30 min. After centrifugation,the cells around the boundary of the Ficoll-Hapaque were collected andwashed three times with PBS. Each 2×10⁵ PBMC obtained from differentvolunteers was mixed with each other and cultured for 5 days on the96-well plate. And RPMI with 10% of serum was used as a medium.

The isolated cells were cultured for 24 hours in the presence of 1, 10,50 and 100 μM of NAPS, TAPS, and PS to examine the effect ofcytotoxicity, i.e. the degree of proliferation of the cells.

C2-ceramide was used as control.

The effect of cytotoxicity was measured by MTT-assay as shown in FIG. 3.

The results in FIG. 3 show that C2-ceramide did not show thecytotoxicity when it was treated up to 100 μM. However, NAPS, TAPS andPS showed the strong cytotoxicity to PBMC. Particularly, 100 μM of NAPSand 50 μM of TAPS shows the strong cytotocicity.

6. Effects of the Compositions of the Present Invention on theActivation of Th1 Cells Associated with the Induction of Psoriasis.

6-1. Mixed Leukocyte Reaction (MLR)

Whether the composition of the present invention has a suppressiveeffect on the Th1 cells activation which isolate IFN-X, IL-12, etc. byreacting with TNBSO₃ (heptene) was examined.

The mononuclear cells obtained from spleen of mice as described theabove 4 were used for responder cells, and TNBSO3-conjugated cells wereused for stimulator cells. The stimulator cells were obtained asfollows:

The mononuclear cells were adjusted to be 2×10⁷ cells/ml in PBS and thesame volume of 20 mM TNBSO3 was added and incubated at 37° C. for 10 minin the dark state. After incubation, the cells wrer washed three timeswith PBS.

Each 2×10⁵ responder cells and stimulator cells were mixed with eachother and cultured for 5 days on the 96-well plate. And RPMI with 10% ofserum was used as a medium.

The above cells were cultured for 24 hours in the presence of 1, 10, 50and 100 μM of NAPS, TAPS, and PS to examine the proliferation of thecells.

C2-ceramide was used as control.

The suppressive effect on Th1 cells activation was measured by MTT-assayas shown in FIG. 4.

The results in FIG. 4 show that C2-ceramide, NAPS and PS showed asuppressive effect on Th1 cells activation at 50 μM, and TAPS at 10 μM.Accordingly, the results showed that all of the compositions of thepresent invention had a suppressive effect on Th1 cells activation byTNBSO₃.

6-2. Reactions Among Allogenic Cells

Whether the composition of the present invention has a suppressiveeffect on the Th1 cells activation by allogenic cells in immune reationwas examined.

PBMC obtained from different volunteers as described in the above 5 wereused for responder cells and stimulator cells respectively. PBMC forstimulator cells were used after X-ray irradiation (3000 rad) to inhibitthe proliferation of stimulator cells.

Each 2×10⁵ responder cells and stimulator cells were mixed with eachother and cultured for 5 days on the 96-well plate. And RPMI with 10% ofserum was used as a medium.

The above cells were cultured for 24 hours in the presence of 1, 10, 50and 100 μM of NAPS, TAPS, and PS to examine the proliferation of thecells.

C2-ceramide was used as control.

The suppressive effect on Th1 cells activation was measured by MTT-assayas shown in FIG. 5.

The results in FIG. 5 show that while C2-ceramide showed almost nosuppressive effect, NAPS, TAPS and PS showed a suppressive effect on Th1cells activation against allogenic cells at 50 μM, in particular, TAPSand PS suppress completely

Induction of apoptosis of HaCaT cells by the compositions of the presentinvention is observed in the TUNEL-TdT-mediated dUTP nick endlabeling-assay by using in situ cell death detection kit, POD (Enzo,1684817, Boeringer Mannhein).

In the TC chamber (Lab-TEK chamber Slide w/cover Permanox Slide sterile1 well, 177410), 1×10⁶ HaCaT cells are seeded and cultured on the DMEMmedium for longer than 18 hours and treated with phytosphingosinederivatives. The cells are fixed 24 hours after treating with drugs, andthe endogenous peroxidase is blocked with a blocking solution (3% H₂O₂in MeOH), and permeation of cells is increased by using apermeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate).Then apoptotic cells are labeled by using the mixture of tunnel reactionand are colored by using the DAB substrate (DAKO, K3465). Colored cellsare observed by using a microscope. The results of measurement are shownin FIGS. 6 and 7.

FIG. 6 are diagrams showing the results of the tunnel assay performed bytreating 10 μM and 30 μM phytosphingosine derivatives for 24 hours. Allof phytosphingosine derivatives cause cell death, and particularly, NAPSand TAPS show the most significant effect of cell death.

FIG. 7 is a diagram showing the effect of inducing apoptosis of NAPS andTAPS at a concentration of 30 μM in a time dependent manner. It is seenthat apoptosis is induced 4 hours after treating NAPS and TAPS to have aconcentration of 30 μM.

The effects of the compositions of the present invention on the cellcycle are reviewed.

In the flow cytometric analysis, the cells are treated with trypsinaccording to each time interval, collected and washed with PBS. They arefixed by using 80% ethanol, and propidium iodide and RNase are added tothem. Thereafter, the cell cycle is analyzed by using a flow cytometer.The results are shown in FIG. 8.

As shown in FIG. 8, the trend is shown to be that S phase is reduced upto 12 hours after treating with TAPS and G₂/M phase is increasedrelatively. Thereafter, the trend is shown to be that G₂/M phase isreduced continuously up to 24 hours and sub G₁ phase is increasedrelatively. Sub G₁ phase refers to apoptotic cells, and it is seen thatinduction of apoptosis by phytosphingosine derivatives is increasedrapidly after 24 hours. It may be viewed that such increase in thenumber of apoptotic cells comes from the cells in G₂/M phase of whichdistribution is reduced at the same time interval (The increase in thenumber of cells in G₁ phase after 24 hours is the result of analysis ofliving cells except for apoptotic cells.).

Next, the cell cycle is observed according to the immunofluorescencemethod. In order to look into from which step among G₂/M phase theinduction of apoptosis by TAPS is induced, mitosis is observed by usingβ-tubulin.

HaCaT cells are cultured on a dish with a cover slip and treated withTAPS (30 μM). The cells are washed with PBS at each time interval, fixedwith 5% paraformaldehyde for 15 minutes, and reacted with β-tubulinantibody (1:100) for 1 hour. They were washed with PBS and reacted withCy3-conjugated anti-mouse IgG secondarily. For DNA staining, 0.3 μg/mlof DAPI (Sigma) is added to the cells, which are observed by using afluorescent microscope. The results are shown in FIG. 9.

As shown in FIG. 9, more cells with chromosomes condensed are observed24 hours after treating with TAPS than 12 hours after. Therefore, it isseen that the mechanism of apoptosis occurs during the metaphase ofmitosis.

In order to study the synergistic effect in the cytotoxicity when thecompositions of the present invention are administered in combinationwith Vit D₃ or calcipotriol, the cell viability is analyzed by MTT assaymethod in HaCaT cells human keratinocyte cell line.

To HaCaT cells, 1 μM of Vit D₃ or calcipotriol, or 10 μM each of NAPSand TAPS, is administered singly or in combination with each other, andthey are cultured for 24 hours. And the effects of these drugs on thecytotoxicity according to the single or combined administration areobserved by MTT assay. C2-ceramide is used for comparison. The resultsare shown in Table 4. TABLE 4 Cell viability according to combinedadministration of the compositions of the present invention with Vit D₃or calcipotriol NAPS TAPS C2-ceramide control (10 μM) (10 μM) (10 μM)Control 100 ± 5  86 ± 2 76 ± 2 78 ± 2 Vit D₃ (1 μM) 81 ± 3 34 ± 4 34 ± 471 ± 9 calcipotriol 77 ± 5 24 ± 3 36 ± 8 70 ± 4 (1 μM)

As shown in Table 4, the administration of 1 μM Vit D₃ or calcipotriolfor 24 hours shows the toxicity of 19% or 23%, respectively. Whilesingle administration of 10 μM NAPS shows the cytotoxicity of 14% and of10 μM TAPS does that of 24%, combined application with Vit D₃ orcalcipotriol shows the cytotoxicity of greater than 66% or 76%,respectively, in case of NAPS, and 66% or 64%, respectively, in case ofTAPS. It is observed that the cytotoxic effect is increasedsignificantly by simultaneous administration of Vit D₃ and calcipotriolor NAPS and TAPS rather than by their single administration.

In order to study the synergistic effect in the cytotoxicity by thecotreatment with administration of the compositions of the presentinvention and with UVB irradiation, the cell viability is analyzed byMTT assay method in HaCaT cells.

On a 100-mm dish, 1×10⁶ HaCaT cells are seeded and cultured in DMEMcontaining 10% fetal bovine serum, 100 units/ml penicillin, and 100μg/ml streptomycin for 48 hours. They are treated with trypsin, and1-2×10⁴ cells per well are seeded on a 96-well plate again by using aserum-free medium, which is removed after about 3 hours. HaCaT cells arewashed with PBS three times, and 3.5 ml of PBS per plate is added. It isthen irradiated with 200 J/m² of UVB. After irradiation, the medium isreplaced with a DMEM medium. Then it is treated with thephytosphingosine derivatives of the present invention, and cultured for24 hours. After 2 mg/ml of the MTT reagent is added, it is cultured for4 hours. The medium is removed completely, the cells are suspended inDMSO, and O.D. is measured at 540 nm. The results of measurement areshown in Table 5 as follows: TABLE 5 Cell viability according tocombined processing of administration of the compositions of the presentinvention and irradiation of UVB UVB + UVB + UVB + PS UVB + PS PS-HClPS-HCl NAPS NAPS TAPS TAPS Con 100 ± 2.4  100 ± 2.4  100 ± 5.2  100 ±5.2  100 ± 9.7  100 ± 6.4  100 ± 8.5  100 ± 8.5  UVB — 38 ± 3.3 — 39 ±3.5 — 30 ± 6.6 — 36 ± 8.0 (200 J/m²)  1 μM 133 ± 3.4  38 ± 7.9 83 ± 7.939 ± 4.5 130 ± 10.0 27 ± 5.8 111 ± 7.0  27 ± 7.1  3 μM 93 ± 5.5 30 ± 3.3 78 ± 10.0 24 ± 2.0 117 ± 9.7  17 ± 1.6  97 ± 10.0 27 ± 3.9  5 μM  85 ±10.6 28 ± 2.3 78 ± 6.7 25 ± 1.9 110 ± 14.0 11 ± 1.3 83 ± 8.0 16 ± 3.2 10μM 71 ± 7.6  3 ± 0.9 75 ± 8.2  2 ± 0.5  67 ± 12.0  1 ± 0.1 43 ± 4.0  2 ±0.1

As shown in Table 5, single irradiation of UVB shows the cytotoxicity ofabout 61-70%, treating with only 10-μM NAPS shows that of 33%, andtreating with only 10-μM TAPS shows that of 57%. The cytotoxic effect isshown to be about 98% or greater in case of the combined use of theadministration of NAPS or TAPS and irradiation of UVB.

Therefore, in case of the cotreatment of the administration of NAPS orTAPS composition and irradiation of UVB, it is observed that thecytotoxic effect is increased significantly compared to the case ofsingle treatment of them.

In the subsequent study, genes and proteins involved in induction ofapoptosis by the compositions of the present invention are confirmed.

Firstly, in order to look into the genes involved in apoptosis inducedby the compositions of the present invention, TAPS is treated at thedose of 30 μM for 24 hours, from which mRNA is isolated. Then it isobserved by using an apoptosis array kit.

The labeled cDNA is synthesized by using apoptosis-specific primersoffered by Human Apoptosis Expression Assay (R&D System, Minneapolis,Minn.), which is used for a probe in incubation with 2 μg of the totalRNA at 65° C. overnight. It is then washed with Washing Solution I(0.5×SSPE, 1% (w/v) SDA) three times at a room temperature and withWashing Solution II (0.1% SSPE, 10% (w/v) SDS) for 1 hour at 65° C. Thewashed membranes are exposed to X-ray films at 70° C., and developed.The results are shown in FIG. 10.

As shown in FIG. 10, as to apoptosis-related genes, the expression ofCOX-2 and PIN genes is increased, survivin, which is apoptosissuppressor, is reduced, and Mcl-1 and Bcl-10, which are Bcl-2-relatedgenes, are increased. Also, IL-1β among cytokines is increased, p21which is a cell cycle regulator is increased, but p53 is decreased.

In the confirmation of apoptosis-related proteins, the immunoblotanalysis is performed in order to look into the effects of thecompositions of the present invention on the expression of caspase-3,p53, and Chk1 proteins related to the induction of apoptosis.

HaCaT cells are cultured on a 100-mm dish and collected at theindicative time. The proteins are extracted by adding 500 μl of RIPAlysis buffer (1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS, 0.15 MSDS, 0.01 M sodium phosphate, pH 7.2, 2 mM EDTA, 50 mM sodium fluorideand 0.2 mM sodium vanadate), and the concentration is measured by usingthe Bradford method. 1

Extracted proteins is electrophoresed on 12% polyacrylamide gel, andthey are transferred to the nitrocellulose membrane. The membraneblocked with 5% non-fat milk is reacted with the primary antibodies(caspase-3, p53, and Chk1), washed, and reacted with the secondaryHRP-conjugated antibody. The intensity of proteins is analyzed by usingthe ECL kit. The results are shown in FIGS. 11, 12, and 13.

As shown in FIG. 11, according to the results of observation of theinduction of caspase-3 by NAPS, TAPS, and C2-ceramide at the indicativetime, the cleavage to the active caspase-3 is increased significantly 30minutes after NAPS treatment and shows 6 times greater expression ofinduction to the maximum within 3 hours, and the increased activecaspase-3 is returned to the level of the control group within 24 hoursafter it is maintained for 12 hours.

On the other hand, although TAPS begins to increase active caspase-3production late compared to NAPS does, 5 folds greater expression ofinduction to the maximum is shown within 3 hours after the drugs aretreated, and its increase is maintained for 24 hours.

Also, C2-ceramide increases production of active caspase-3 to themaximum within 3 hours after the drugs are treated, but the increase issignificantly weak compared to NAPS and TAPS (increase by 2.5 folds).Through the above results, it is seen that phytosphingosine derivativeshave a superior ability to induce apoptosis, and is more superior thanC2-ceramide known conventionally in the efficacy related to theinduction of apoptosis.

As shown in FIGS. 12 and 13, p53 and Chk1 proteins show the tendency ofrapid reduction at 24 hour after treated which apoptosis occurs althoughthey are not changed greatly by treatment with TAPS at 8-hour. Theseresults mean that p53 and Chk1 paths are involved in the induction ofapoptosis by ceramide. It is seen, particularly, that Chk1 is a proteininvolved in the inhibition of G₂/M phase by the damage of DNA.

In the next study, the effects of treatment of psoriasis by thecompositions of the present invention are reviewed. NAPS, which is aneffective component of the compositions of the present invention, ismade to have a concentration of 0.5%, and a cream formulation ismanufactured at a composition ratio of the following components and usedfor a reagent: NAPS 0.5 g Stearic acid 1.0 g Cetyl alcohol 1.4 g Stearylalcohol 1.4 g Glyceryl monostearate 2.0 g Solbitan monostearate 0.2 gMethyl paraben 0.2 g Propyl paraben 0.1 g Mineral oil 10.0 g Caprylic/capric acid 3.0 g MDF (meadow foam seed oil) 3.0 g Dimethicone(methyl polysiloxane) 0.5 g Solbitan cesquiolate 0.2 g Twin 60 1.2 gDisodium EDTA 0.02 g  Glycerin 3.0 g Trimethanolamine 0.2 g Sepigel 3050.5 g Zermol 115 0.2 g Purified water 71.28 g 

Four each 20- to 40-year-old male and female patients are in theexperimental group. The reagent manufactured in the above is applied tothe psoriatic lesions of 8 patients and the changes are observed.Improved symptoms of each psoriatic lesions are shown in FIG. 14 (Case1: elbow, Case 2: forehead, Case 3: knee). It is confirmed that 7 among8 patients have considerably improved symptoms 5-7 days afterapplication of cream preparations of the compositions of the presentinvention. Therefore, it is seen that the compositions of the presentinvention are effective for the treatment of psoriasis.

Six kinds of phytosphingosine derivatives used for the present inventionshow superior effects in the cytotoxicity and induction of apoptosis.Accordingly, the compositions of the present invention containing thesephytosphingosine derivatives as effective components are useful for theprevention or treatment of various skin diseases, various tumors,various cancers, etc. that may be prevented or cured by the induction ofapoptosis in living bodies.

While the invention has been described in terms of a single preferredembodiment, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims.

1. A pharmaceutical composition for the induction of apoptosiscontaining phytosphingosine derivatives as effective components.
 2. Thepharmaceutical composition for the induction of apoptosis of claim 1,wherein said phytosphingosine derivatives are one or more compoundsselected from a group comprised of phytosphingosine,phytosphingosine-HCl, C6-phytosphingosine, CLA-phytosphingosine,tetraacetyl phytosphingosine, and N-acetyl phytosphingosine.
 3. Thepharmaceutical composition for the induction of apoptosis of claim 1,wherein Vitamin D₃ or calcipotriol is contained additionally in saidphytosphingosine derivatives.
 4. The pharmaceutical composition of claim1, which is useful for the prevention or treatment of various skindiseases, various tumors, various cancers, and other diseases that maybe prevented or cured by the induction of the activity of apoptosis inliving bodies.
 5. The pharmaceutical composition for the induction ofapoptosis of claim 4, which is useful for the prevention or treatment ofabnormal skin diseases such as eczema, psoriasis, ichthyosis, andothers; skin diseases such as atopic dermatitis, dermatitis, itching,microbism, acne, wound, and others; abnormal skin diseases induced by along-time exposure to UV light and dermal aging; and skin cancer andothers.
 6. The pharmaceutical composition for the induction of apoptosisof claim 5, which is useful for the prevention or treatment of abnormalskin diseases such as psoriasis, ichthyosis, and others and abnormalskin diseases induced by a long-time exposure to UV light and dermalaging.
 7. The pharmaceutical composition for the induction of apoptosisof claim 5, which is useful for the prevention or treatment of skincancer.
 8. A cosmetic composition for the induction of apoptosiscontaining phytosphingosine derivatives as effective components.
 9. Thecosmetic composition for the induction of apoptosis of claim 8, whereinsaid phytosphingosine derivatives are one or more compounds selectedfrom a group comprised of phytosphingosine, phytosphingosine-HCl,C6-phytosphingosine, CLA-phytosphingosine, tetraacetyl phytosphingosine,and N-acetyl phytosphingosine.
 10. The cosmetic composition for theinduction of apoptosis of claim 8, wherein vitamin D₃ or calcipotriol iscontained additionally in said phytosphingosine derivatives.
 11. Thecosmetic composition for the induction of apoptosis of claim 8, which isuseful for the prevention or treatment of various skin diseases, varioustumors, various cancers, and other diseases that may be prevented orcured by the induction of the activity of apoptosis in living bodies.12. The cosmetic composition for the induction of apoptosis of claim 12,which is useful for the prevention or treatment of abnormal skindiseases such as eczema, psoriasis, ichthyotis, and others; skindiseases such as atopic dermatitis, dermatitis, itching, microbism,acne, wound, and others; abnormal skin diseases induced by a long-timeexposure to UV light and dermal aging; and skin cancer and others. 13.The cosmetic composition for the induction of apoptosis of claim 12,which is useful for the prevention or treatment of abnormal skindiseases such as psoriasis, ichthyosis, and others; and abnormal skindiseases induced by a long-time exposure to UV light and dermal aging.14. The cosmetic composition for the induction of apoptosis of claim 12,which is useful for the treatment of skin cancer.
 15. A method ofprevention or treatment of various skin diseases, various tumors,various cancers, and other diseases that may be prevented or cured bythe induction of the activity of apoptosis in living bodies, comprisingthe steps of the administration of compositions for the induction ofapoptosis containing phytosphingosine derivatives as effectivecomponents and of the irradiation of UVB to a psoriatic lesion.
 16. Themethod of prevention or treatment of various skin diseases, varioustumors, various cancers, and other diseases of claim 15, wherein saidphytosphingosine derivatives are one or more compounds selected from agroup comprised of phytosphingosine, phytosphingosine-HCl,C6-phytosphingosine, CLA-phytosphingosine, tetraacetyl phytosphingosine,and N-acetyl phytosphingosine.
 17. The method of prevention or treatmentof claim 15, wherein abnormal skin diseases such as eczema, psoriasis,ichthyotis, and others; skin diseases such as atopic dermatitis,dermatitis, itching, microbism, acne, wound, and others; abnormal skindiseases induced by a long-time exposure to UV light and dermal aging;and skin cancer and others are prevented or treated.
 18. The method ofprevention or treatment of claim 17, wherein abnormal skin diseases suchas psoriasis, ichthyosis, and others and abnormal skin diseases inducedby a long-time exposure to UV light and dermal aging are prevented ortreated.
 19. The method of prevention or treatment of claim 17, whereinskin cancer is prevented or treated.